V?14i NKT cells are a distinct sublineage of T lymphocytes with a unique developmental pathway, and a surprising ability to influence many different types of immune responses. Their influence can be unpredictable, however, and they may exhibit opposing effects on tumor growth, inflammation, and other responses. In this application, we will use biochemical and cellular immunology methods to determine how the affinity of interaction of the TCR of V?14i NKT cells with different glycolipids, including natural antigens and ligands, influences their differentiation. By analyzing agonists of different strength and by mutating CD1d amino acids, we will determine if the development of V?14i NKT cells requires a strong signal and how changes in signal strength influence the behavior and phenotype, as well as the numbers of these cells. We also will determine if CDS acts as a co-receptor for CD1d-mediated antigen recognition. Analogs of ?GalCer, which strongly activate V?14i NKT cells, have been reported to promote Th1 (C-glycoside) or Th2 (OCH) cytokine synthesis. We will determine if this is related to the stability of their binding to CD1d, the TCR affinity for the antigen, length of the stimulus, or the ARC that preferentially present different antigens. We also will determine if downstream effects of V?14i NKT cell activation, such as IL-12 release and changes in dendritic cells are responsible for cytokine polarization. The specificity of V?14i NKT cells is conserved in humans, and stimulation of V?i NKT cells is being tested as a treatment for tumors and hepatitis infection. The experiments here will analyze how the stimulation of the TCR relates to the divergent outcomes that follow their stimulation. This knowledge may lead to immune based therapies that specifically target these cells for treatment of cancer and other diseases.